Analysis of 9703 expressed sequence tags in cold acclimated oat

Winter oat, Avena sativa cv. Gerald, was cold acclimated at +4oC for 4, 6, 8 and 32 hours. Total RNA was prepared from these plants, pooled and a cDNA library was constructed. From this library 9792 expressed sequence tags (EST) were sequenced. The average sequence length after vector clipping was 626 bp and the longest sequences were over 900 bp. Clustering and assembly of the these 9792 EST sequences resulted in a set of 4543 sequences. Clustering, assembly and filtering of these sequences resulted in a set of 2866 unique transcripts, denoted the "UniGene" set. Homology searches on publicly available sequence data allowed the assignment of a tentative function to 1622 (57.57%) of these transcripts. Out of the remaining 1246 unclassified sequences, 494 appeared to be oat specific since they lacked any significant sequence similarity (E-values > 1e-10 after BlastX search) to any sequence presently available in the public databases. Genes active in photsynthesis were most commonly found, but genes involved in metabolism, signal transduction and abiotic stress were also well represented. Interestingly, 398 sequences displayed strong homologies (E-values ? 1e-10 after BlastX search) to genes previously reported to be involved in cold stress related processes. Of particular importance for the regulation of coldacclimation are cold induced transcription factors. We found 47 such genes, including 4 CBF transcription factor sequences, a known cold regulatory gene class (see other abstract by Bräutigam et al). The set of 4543 clustered and assembled sequences (in total 5.3 Mb) was searched for microsatellites (SSRs) and 596 di- to pentanucleotide SSRs were found. 83% of these SSRs occurred in non-coding sequences. Work is now in progress to identify the particular SSRs from this collection that give reliable PCR products and are polymorphic. The best SSR markers will then be mapped to the oat genome and linked to valuable genetic traits like lipid biosynthesis, antioxidant activity and cold hardiness.